A Highly Efficient Method to Generate Chimeric Soybean Plant with Transgenic Hairy Roots

doi:10.11983/CBB23021

Abstract

Abstract: Chimeric soybean plants with transgenic hairy roots is very important for soybean functional genomics. In this study, we used three soybean genotypes to compare their hairy root induction rate and plant survival rate under different co-cultivation conditions. Our results showed that co-culturing the explants infected by Agrobacterium rhizogenes for 1 d under dark conditions was an effective strategy to induce hairy roots. We also found that removing the adventitious roots (AR) at hypocotyl significantly increased number of hairy roots, enhanced their growth and subsequently improved the positive rate of transgenic hairy roots. Furthermore, we found that the inoculation with rhizobium at 14 d of induction was able to enhance the contact between the bacteria and the transgenic hairy roots at early growth stages, and thus improved the soybean’s nodulation efficiency. Taken together, we successfully established a simple and efficient method to generate chimeric soybean plants with transgenic hairy roots. This method can be widely used in soybean gene functional studies.

Key words: soybean, hairy root, chimeric plant, nodulation, optimization

Jiaxin Chen, Hao Mei, Caixiang Huang, Zongyuan Liang, Yitong Quan, Dongpeng Li, Buweimaieryemu·Saimaiti , Xinxin Li, Hong Liao. A Highly Efficient Method to Generate Chimeric Soybean Plant with Transgenic Hairy Roots[J]. Chinese Bulletin of Botany, 2024, 59(1): 89-98.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB23021

https://www.chinbullbotany.com/EN/Y2024/V59/I1/89

Figures/Tables 6

Figure 1 Procedure of generating chimeric soybean plants with Agrobacterium rhizogenes induced hypocotyl transgenic hairy roots (A) Soybean seedlings at 4 d post germination (bar=2 cm); (B) Hypocotyl was cut to form wound site with an angle of 45° (bar=1 cm); (C) A. rhizogenes were collected in petri dishes (bar=1 cm); (D) Apply bacteria to the wound (bar=1 cm); (E) Co-cultivation (bar=1 cm); (F) Inoculated explants were planted into vermiculite (bar=5 cm); (G) Callus came out after 10 d of cultivation (bar=1 cm); (H) Hairy roots emerged at 14 d post induction (bar=1 cm); (I) Growth performance of hairy roots after 20 d of growth (bar=2 cm); (J) Partial enlarged view of image I (bar=2 cm)

Table 1 Comparison of hairy roots (HR) generated from different soybean genotypes under light/dark conditions

Treatments	Genotype	Co-cultivation time (d)	Efficiency of induction (%)	Number of chimeric plant with HR	Surviving rate (%)	Number of surviving plants	Total number of plants
Dark	Ws82	0	87.0	20	76.7	23	30
		1	96.2	25	86.7	26	30
		3	89.7	26	96.7	29	30
	HN66	0	87.5	21	80.0	24	30
		1	95.7	22	76.7	23	30
		3	83.3	15	60.0	18	30
	BX10	0	93.3	14	75.0	15	20
		1	94.7	18	95.0	19	20
		3	82.4	14	85.0	17	20
Light (100 µmol∙m^-2∙s^-1)	Ws82	0	82.4	21	76.7	23	30
Light (100 µmol∙m^-2∙s^-1)		1	91.3	21	76.7	23	30
		3	68.2	15	73.3	22	30
	HN66	0	90.0	18	66.7	20	30
		1	83.3	20	80.0	24	30
		3	90.5	19	70.0	21	30
	BX10	0	86.7	13	75.0	15	20
		1	83.3	15	90.0	18	20
		3	80.0	12	75.0	15	20

Figure 2 Effect of cleaning adventitious root at hypocotyl on induction of hairy roots (A) Comparison of hairy roots between non-clean and clean adventitious root condition (bars=1 cm); (B) Number of lateral roots per hairy root; (C) Root length; (D) Root surface area; (E) Average root diameter. Data are means±SD from 25 hairy roots. *** indicate significant differences at 0.001 level. AR: Adventitious root; HR: Hairy root

Figure 3 Effect of cleaning adventitious root at hypocotyl on positive rate of transgenic hairy roots (A) GUS staining of hairy roots under non-clean and clean adventitious roots conditions, the oval and arrows indicate non-transgenic hairy roots (bars=1 cm); (B) Total number of hairy roots generated from each explant; (C) Number of transgenic hairy roots per explant; (D) Efficiency of transgenic hairy roots. Data are means±SD from 14 explants. * and *** indicate significant differences at 0.05 and 0.001 levels, respectively.

Figure 4 Effect of cleaning adventitious root on nodulation of hairy roots (A), (D) Soybean chimeric plants without (A) or with (D) cleaning adventitious root (bars=2 cm); (B), (E) Partial enlarged view of image A and D in box (bars=1 cm); (C), (F) GUS staining of hairy roots (bars=1 cm). The red arrows indicate nodules on hairy roots. AR is the same as shown in Figure 2.

Table 2 Effect of non-clean (NCAR) and clean (CAR) adventitious root on nodule number in transgenic hairy roots

Treatments	Chimeric plants	Hairy roots	Nodule number
NCAR	1^st	1^st	3
		2^nd	4
		3^rd	2
	2^nd	1^st	2
		2^nd	1
		3^rd	3
	3^rd	1^st	4
		2^nd	2
		3^rd	1
Average			2.4
CAR	1^st	1^st	7
		2^nd	6
		3^rd	8
	2^nd	1^st	10
		2^nd	8
		3^rd	6
	3^rd	1^st	5
		2^nd	7
		3^rd	9
Average			7.3***

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